This application is the U.S. national phase of PCT/AT98/00020 filed Jan. 1, 1998, which claims priority from the Austrian application A 176/97 filed Feb. 4, 1997.
The invention relates to a method of recovering highly purified vWF or a factor VIII/vWF-complex from a biological starting material by means of collagen-affinity chromatography, as well as to a stable preparation containing highly purified vWF or factor VIII/vWF-complex.
vWF is of particular importance in physiological hemostasis. Besides its function as a carrier protein for factor VIII, this plasma protein above all is necessary for the adhesion of thrombocytes to the damaged endothelium/subendothelium as well as for the aggregation of the thrombocytes under shearing stress conditions. In the first stage of primary hemostasis, the adhesion, vWF acts as a binding element between specific receptors of the thrombocyte surface, such as gpIb, gpIIb/IIIa-complex, and components of the endothelium, e.g. collagen.
According to EP 0 503 991 A, vWF is obtained in purified form from pre-purified plasma by the combination of three chromatographic purification steps. These chromatographic purification steps include a two-fold chromatography on an ion exchanger and, as a third step, an affinity chromatographic purification on gelatin sepharose. It has been shown that in gelatin sepharose chromatography, contaminating proteins, such as, e.g., fibronectin, are bound, and vWF can be obtained in the eluate.
The purification of vWF by a fractogel-EMD-TMAE chromatography with subsequent chromatography on heparin-fractogel-EMD has been described by Fischer et al. (Thromb. Res. 84 (1) (1996), 55-66).
Collagen is a physiological binding partner of vWF. Studies by Kessler et al. (Blood 63 (6) (1984), 1291-1298) have shown that the complex of vWF and factor VIII binds to collagen fibrillae, yet, however, not to denatured collagen. This result has been confirmed many times, e.g. by Santoro et al. (Collagen Rel. Res. 2 (1982), 31-43), who found out that only native collagen, yet not denatured collagen, constitutes a binding partner for vWF (cf. also Santoro, Thromb. Res. 21 (1981), 689-693, and BBA 756 (1983), 123-126; confirmed also by EP 0 503 991 A, since vWF passed the gelatine sepharose column without hindrance). It has been shown that the high molecular forms of vWF were bound by limiting low collagen concentrations. High and medium molecular forms of vWF were also bound by non-limiting high collagen concentrations, whereas the low molecular vWF fraction was not even bound at high collagen concentrations (Santoro (1983)).
This was also the reason for the fact that in assaying methods which were based on vWF collagen binding, always native collagen was used and the nativity thereof was also retained in the assay, in that denaturing conditions or strong or covalent bonds of the collagen to a solid carrier were not described in these binding assays (Brown et al., Thromb. Res. 43 (1985), 303-311).
Although the binding of collagen to vWF has been described in detail, and it has even been suggested to include this binding of vWF to native collagen in a vWF purification method (Santoro et al. (1982)), hitherto no suitable preparative method based on collagen-vWF-binding has been provided. On the one hand, this has been due to the fact that chromatographic materials based on collagen (such as, e.g., gelatine sepharose) have proven unsuitable for vWF binding, and, on the other hand, that collagen fibrillae are not suitable for the preparative recovery of vWF (cf. Santoro et al. (1982)).
It is the object of the present invention to provide an improved method of purifying and recovering vWF. The method comprises absorbing vWF or factor VIII/vWF complex to immobilized collagen and recovering the adsorbed vWF or factor VIII/vWF complex by eluting the adsorbed material after removing non-adsorbed material. The method according to the preset invention results in a high proportion of physiologically active vWF and can be performed on an industrial scale.
According to the invention, this object is achieved by a method of the initially defined way which comprises the following steps:
adsorbing the vWF from the starting material on avid collagen immobilized on a carrier,
separating the non-adsorbed portion and, optionally, washing the carrier,
eluting the vWF from immobilized collagen, and
recovering the purified vWF.